The focus of this project is the structure determination of Na, K- ATPase, an integral membrane protein that constitutes the Na, K pump in the plasma membrane of animal cells. Three-dimensional (3-D) reconstruction from tilted views of negatively stained sheets has revealed the structure of the cytoplasmic component of Na, K-ATPase At 2.5nm resolution. The domains corresponding to the two subunits of the enzyme have been identified in the structure. This information will be extended by two new approaches: electron microscopy and image processing of frozen hydrated specimens of the sheets will reveal structural organization of both the intra- and the extra-membrane regions of the enzyme; freeze-drying and high resolution metal shadowing of the crystalline sheets and surface reconstruction will provide information about the exoplasmic surface that is not available in the current map. Furthermore, it will provide a baseline for interpreting subsequent images of the crystalline enzyme after biochemical modification. Attempts will be made to extend the structural studies to higher resolution by forming better-ordered and larger crystalline sheets. The carbohydrate moiety of the beta subunit will be removed. Crystallization of the latter should result in the formation of sheets with greater degree of order. Additionally, comparison of the modified structure with that of the intact enzyme will give further evidence for the location of the beta subunit. Solubilization of the enzyme in nonionic detergents and reconstitution with controlled amounts of phospholipids would also lead to the formation of better-ordered and larger crystalline sheets. An ultimate goal for the electron microscopy work is obtaining a 3-D structure of the enzyme at approximately 1.0nm resolution. This would be sufficient to resolve structures such as helices and channels. By correlating these results with the information from the location of the two subunits, and the sequence the structure of various domains can be related to their role in the transport process. In parallel with the above studies, the structure of H, K-ATPase will be determined by method similar to those used for Na, K- ATPase. H, K-ATPase constitutes the gastric pump that is responsible for the secretion of acid into the stomach. It is closely related to Na, K-ATPase and the resulting structural similarities and differences could shed light on the mechanism of ion transport by these important biological systems.
| Mohraz, M; Arystarkhova, E; Sweadner, K J (1994) Immunoelectron microscopy of epitopes on Na,K-ATPase catalytic subunit. Implications for the transmembrane organization of the C-terminal domain. J Biol Chem 269:2929-36 |
| Mohraz, M; Smith, P R (1988) Three-dimensional structure of Na,K-ATPase and a model for the oligomeric form and the mechanism of the Na,K pump. Prog Clin Biol Res 268A:99-106 |
| Mohraz, M; Simpson, M V; Smith, P R (1987) The three-dimensional structure of the Na,K-ATPase from electron microscopy. J Cell Biol 105:1-8 |
| Mohraz, M; Yee, M; Smith, P R (1986) Structural studies of Na,K-ATPase. Ann N Y Acad Sci 483:131-9 |
| Mohraz, M; Yee, M; Smith, P R (1985) Novel crystalline sheets of Na,K-ATPase induced by phospholipase A2. J Ultrastruct Res 93:17-26 |
